Flaviviridae Viruses
The Flaviviridae family of viruses comprises at least three distinct genera: pestiviruses, which cause disease in cattle and pigs; flaviviruses, which are the primary cause of diseases such as dengue fever and yellow fever; and hepaciviruses such as hepatitis C (HCV). The flavivirus genus includes more than 68 members separated into groups on the basis of serological relatedness (Calisher et al., J. Gen. Virol, 1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitis and hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that are associated with human disease include Dengue virus, hemorrhagic fever viruses such as Lassa, Ebola, and yellow fever virus, shock syndrome, and Japanese encephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6, 251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., New Eng. J. Med., 1988, 319, 641-643).
The pestivirus genus includes bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV, also called hog cholera virus) and border disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res. 1992, 41, 53-98). Pestivirus infections of domesticated livestock (cattle, pigs and sheep) cause significant economic losses worldwide. BVDV causes mucosal disease in cattle and is of significant economic importance to the livestock industry (Meyers, G. and Thiel, H.-J., Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv. Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been as extensively characterized as the animal pestiviruses. However, serological surveys indicate considerable pestivirus exposure in humans.
Pestiviruses and hepaciviruses are closely related virus groups within the Flaviviridae family. Other closely related viruses in this family include the GB virus A, GB virus A-like agents, GB virus-B and GB virus-C (also called hepatitis G virus, HGV). The hepacivirus group (hepatitis C virus; HCV) consists of a number of closely related but genotypically distinguishable viruses that infect humans. There are approximately 6 HCV genotypes and more than 50 subtypes. Due to the similarities between pestiviruses and hepaciviruses, combined with the poor ability of hepaciviruses to grow efficiently in cell culture, bovine viral diarrhea virus (BVDV) is often used as a surrogate to study the HCV virus.
The genetic organization of pestiviruses and hepaciviruses is very similar. These positive stranded RNA viruses possess a single large open reading frame (ORF) encoding all the viral proteins necessary for virus replication. These proteins are expressed as a polyprotein that is co- and post-translationally processed by both cellular and virus-encoded proteinases to yield the mature viral proteins. The viral proteins responsible for the replication of the viral genome RNA are located within approximately the carboxy-terminal. Two-thirds of the ORF are termed nonstructural (NS) proteins. The genetic organization and polyprotein processing of the nonstructural protein portion of the ORF for pestiviruses and hepaciviruses is very similar. For both the pestiviruses and hepaciviruses, the mature nonstructural (NS) proteins, in sequential order from the amino-terminus of the nonstructural protein coding region to the carboxy-terminus of the ORF, consist of p7, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5A, and NS5B.
The NS proteins of pestiviruses and hepaciviruses share sequence domains that are characteristic of specific protein functions. For example, the NS1 glycoprotein is a cell-surface protein that is translocated into the ER lumen. NS1 was characterized initially as soluble complement-fixing antigen found in sera and tissues of infected animals, and now is known to elicit humoral immune responses in its extracellular form. Antibodies to NS1 may be used to confer passive immunity to certain pestiviruses and flaviviruses. NS1 has been implicated in the process of RNA replication where it is believed to have a functional role in the cytoplasmic processing of RNA. NS2A is a small (approximately 22 kd) protein of unknown function. Studies suggest that it binds to NS3 and NS5, and so may be a recruiter of RNA templates to membrane-bound replicase. NS2B also is a small (about 14 kd) protein that is membrane-associated, and is a required cofactor for the serine protease function of NS3, with which it forms a complex.
The NS3 proteins of viruses in both groups are large (about 70 kd), membrane-associated proteins that possess amino acid sequence motifs characteristic of serine proteinases and of helicases (Gorbalenya et al. (1988) Nature 333:22; Bazan and Fletterick (1989) Virology 171:637-639; Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897). Thus, the NS3 proteins have enzymatic activity needed for processing polyproteins for RNA replication. The C-terminal end of the NS3 proteins have an RNA triphosphotase activity that appears to modify the 5′ end of the genome prior to 5′-cap addition by guanylyltransferase.
NS4A and NS4B are membrane-associated, small (about 16 kd and about 27 kd, respectively), hydrophobic proteins that appear to function in RNA replication by anchoring replicase components to cellular membranes (Fields, Virology, 4th Edition, 2001, p. 1001).
The NS5 proteins are the largest (about 103 kd) and most conserved, with sequence homology to other (+)-stranded RNA viruses. It also plays a pivotal role in viral replication. The NS5B proteins of pestiviruses and hepaciviruses are the enzymes necessary for synthesis of the negative-stranded RNA intermediate that is complementary to the viral genome, and of the positive-stranded RNA that is complementary to the negative-stranded RNA intermediate. The NS5B gene product has Gly-Asp-Asp (GDD) as a hallmark sequence, which it shares with reverse transcriptases and other viral polymerases and which is predictive of RNA dependent RNA polymerase (RdRP) activity (DeFrancesco et al., Antiviral Research, 2003, 58:1-16). Interestingly, it was found that the NS5B C-terminal 21 residue long hydrophobic tail is needed to target NS5B to the ER membrane, but its removal has no other effect and, in fact, leads to increased enzymatic solubility and activity (Tomei et al., J. Gen. Virol., 2000, 81:759-767; Lohmann et al., J. Virol., 1997, 71:8416-28; Ferrari et al., J. Virol., 1999, 73:1649-54).
The NS5B enzyme products have the motifs characteristic of RNA-directed RNA polymerases, and in addition, share homology with methyltransferase enzymes that are involved in RNA cap formation (Koonin, E. V. and Dolja, V. V. (1993) Crit. Rev. Biochem. Molec. Biol. 28:375-430; Behrens et al. (1996) EMBO J. 15:12-22; Lchmann et al. (1997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys. Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al. (1998) J. Virol. 72.9365-9369). The unliganded crystal structure of NS5B shows the unique structural feature of folding in a classic “right hand” shape, in which fingers, palm and thumb subdomains can be recognized (a feature it shares with other polymerases), but differs from other “half-open right hand” polymerases by having a more compact shapes due to two extended loops that span the finger and thumb domains at the top of the active site cavity (DeFrancesco et al. at 9). The finger, thumb and palm subdomains encircle the active site cavity to which the RNA template and NTP substrates have access via two positively charged tunnels (Bressanelli et al., J. Virol., 2002, 76, 3482-92). Finger and thumb domains have strong interactions that limit their ability to change conformation independently of one another, a structural feature shared by other RdRPs. The thumb domain contains a β-hairpin loop that extends toward the cleft of the active site and may play a role in restricting the binding of the template/primer at the enzyme active site (DeFrancesco et al., at 10). Studies are in progress to determine the role of this loop in the initiation mechanism of RNA synthesis (Id.)
Nucleotidyl transfer reaction residues are located in the palm domain and contain the signature GDD motif (DeFrancesco et al., at 9). Palm domain geometry is highly conserved in all polymerases, and has a conserved two-metal-ion catalytic center that is required for catalyzing a phosphory transfer reaction at the polymerase active site.
It is believed that the de novo initiation model of RNA polymerization, rather than a “copy back” mechanism, is utilized by pesti-, flavi- and hepaciviruses. In the de novo initiation model, complementary RNA synthesis is initiated at the 3′-end of the genome by a nucleotide triphosphate rather than a nucleic acid or a protein primer. Purified NS5B is capable of this type of primer-independent action, and the C-terminal β-loop is believed to correctly position the 3′-end of the RNA template by functioning as a gate that retards slippage of the RNA 3′-end through the polymerase active site (Hong et al., Virology, 2001, 285:6-11. Bressanelli et al. reported the structure of NS5B polymerase in complex with nucleotides in which three distinct nucleotide-binding sites were observed in the catalytic center of the HCV RdRP, and the complex exhibited a geometry similar to the de novo initiation complex of phi 6 polymerase (Bressanelli et al., J. Virol., 2002, 76: 3482-92). Thus, de novo initiation occurs and apparently is followed by RNA elongation, termination of polymerization, and release of the new strand. At each of these steps is the opportunity for intervention and inhibition of the viral lifecycle.
The actual roles and functions of the NS proteins of pestiviruses and hepaciviruses in the lifecycle of the viruses are directly analogous. In both cases, the NS3 serine proteinase is responsible for all proteolytic processing of polyprotein precursors downstream of its position in the ORF (Wiskerchen and Collett (1991) Virology 184:341-350; Bartenschlager et al. (1993) J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem. Biophys. Res. Comm. 192:399-406; Grakoui et al. (1993) J. Virol. 67:2832-2843; Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA 90:10583-10587; Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome et al. (1993) J. Virol. 67:4017-4026). The NS4A protein, in both cases, acts as a cofactor with the NS3 serine protease (Bartenschlager et al. (1994) J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68: 3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J. Virol. 71:5312-5322). The NS3 protein of both viruses also functions as a helicase (Kim et al. (1995) Biochem. Biophys. Res. Comm. 215: 160-166; Jin and Peterson (1995) Arch. Biochem. Biophys., 323:47-53; Warrener and Collett (1995) J. Virol. 69:1720-1726). Finally, the NS5B proteins of pestiviruses and hepaciviruses have the predicted RNA-directed RNA polymerases activity (Behrens et al. (1996) EMBO J. 15:12-22; Lchmann et al. (1997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys. Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al. (1998) J. Virol. 72.9365-9369).
Hepatitis C Virus
The hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000). HCV causes a slow growing viral infection and is the major cause of cirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 80-85, (1999); Boyer, N. et al. J. Hepatol. 32:98-112, 2000). An estimated 170 million persons are infected with HCV worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000). Cirrhosis caused by chronic hepatitis C infection accounts for 8,000-12,000 deaths per year in the United States, and HCV infection is the leading indication for liver transplantation.
HCV is known to cause at least 80% of posttransfusion hepatitis and a substantial proportion of sporadic acute hepatitis. Preliminary evidence also implicates HCV in many cases of “idiopathic” chronic hepatitis, “cryptogenic” cirrhosis, and probably hepatocellular carcinoma unrelated to other hepatitis viruses, such as Hepatitis B Virus (HBV). A small proportion of healthy persons appear to be chronic HCV carriers, varying with geography and other epidemiological factors. The numbers may substantially exceed those for HBV, though information is still preliminary; how many of these persons have subclinical chronic liver disease is unclear. (The Merck Manual, ch. 69, p. 901, 16th ed., (1992)).
HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5′ untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation. Translation of the HCV genome is initiated by a cap-independent mechanism known as internal ribosome entry. This mechanism involves the binding of ribosomes to an RNA sequence known as the internal ribosome entry site (IRES). An RNA pseudoknot structure has recently been determined to be an essential structural element of the HCV IRES. Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, E1 and E2.
HCV also encodes two proteinases, a zinc-dependent metalloproteinase encoded by the NS2-NS3 region and a serine proteinase encoded in the NS3 region. These proteinases are required for cleavage of specific regions of the precursor polyprotein into mature peptides: the junction between NS2 and NS3 is autocatalytically cleaved the NS2/NS3 protease, while the remaining junctions are cleaved by the N-terminal serine protease domain of NS3 complexed with NS4A. The NS3 protein contains the NTP-dependent helicase activity that unwinds duplex RNA during replication. The hydrophobic carboxy-terminal 21 amino acids of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase that is essential for viral replication (Fields Virology, Fourth Edition, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., 2001, Chapter 32, pp. 1014-1015). NS5B is known to bind RNAs nonspecifically, and to interact directly with NS3 and NS4A that, in turn, form complexes with NS4B and NS5A (Id @ 1015; Ishido et al., Biochem. Biophys. Res. Commun., 1998; 244:35-40). Certain in vitro experiments using NS5B and guanosine 5′-mono-, di-, and triphosphate as well as 5′-triphosphate of 2′-deoxy- and 2′,3′-dideoxy-guanosine as HCV inhibitors suggest that HCV-RdRP may have a strict specificity for 5′-triphosphates and 2′- and 3′-OH groups (Watanabe et al., U.S. 2002/0055483). Otherwise, the function(s) of the remaining nonstructural proteins, NS4A, NS4B, and NS5A (the amino-terminal half of nonstructural protein 5) remain unknown.
A significant focus of current antiviral research is directed to the development of improved methods of treatment of chronic HCV infections in humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 80-85, (1999)).
Methods to Treat Flaviviridae Infections
The development of new antiviral agents for Flaviviridae infections, especially hepatitis C, is currently underway. Specific inhibitors of HCV-derived enzymes such as protease, helicase, and polymerase inhibitors are being developed. Drugs that inhibit other steps in HCV replication are also in development, for example, drugs that block production of HCV antigens from the RNA (IRES inhibitors), drugs that prevent the normal processing of HCV proteins (inhibitors of glycosylation), drugs that block entry of HCV into cells (by blocking its receptor) and nonspecific cytoprotective agents that block cell injury caused by the virus infection. Further, molecular approaches are also being developed to treat hepatitis C, for example, ribozymes, which are enzymes that break down specific viral RNA molecules, and antisense oligonucleotides, which are small complementary segments of DNA that bind to viral RNA and inhibit viral replication, are under investigation. A number of HCV treatments are reviewed by Bymock et al. in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000) and De Francesco et al. in Antiviral Research, 58: 1-16 (2003).
Idenix Pharmaceuticals, Ltd. discloses branched nucleosides, and their use in the treatment of HCV and flaviviruses and pestiviruses in US Patent Publication Nos. 2003/0050229 A1, 2004/0097461 A1, 2004/0101535 A1, 2003/0060400 A1, 2004/0102414 A1, 2004/0097462 A1, and 2004/0063622 A1 which correspond to International Publication Nos. WO 01/90121 and WO 01/92282. A method for the treatment of hepatitis C infection (and flaviviruses and pestiviruses) in humans and other host animals is disclosed in the Idenix publications that includes administering an effective amount of a biologically active 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides or a pharmaceutically acceptable salt or prodrug thereof, administered either alone or in combination, optionally in a pharmaceutically acceptable carrier. See also U.S. Patent Publication Nos. 2004/0006002 and 2004/0006007 as well as WO 03/026589 and WO 03/026675. Idenix Pharmaceuticals, Ltd. also discloses in US Patent Publication No. 2004/0077587 pharmaceutically acceptable branched nucleoside prodrugs, and their use in the treatment of HCV and flaviviruses and pestiviruses in prodrugs. See also PCT Publication Nos. WO 04/002422, WO 04/002999, and WO 04/003000. Further, Idenix Pharmaceuticals, Ltd. also discloses in WO 04/046331 Flaviviridae mutations caused by biologically active 2′-branched β-D or β-L nucleosides or a pharmaceutically acceptable salt or prodrug thereof.
Biota Inc. discloses various phosphate derivatives of nucleosides, including 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides, for the treatment of hepatitis C infection in International Patent Publication WO 03/072757.
Emory University and the University of Georgia Research Foundation, Inc. (UGARF) discloses the use of 2′-fluoronucleosides for the treatment of HCV in U.S. Pat. No. 6,348,587. See also US Patent Publication No. 2002/0198171 and International Patent Publication WO 99/43691.
BioChem Pharma Inc. (now Shire Biochem, Inc.) discloses the use of various 1,3-dioxolane nucleosides for the treatment of a Flaviviridae infection in U.S. Pat. No. 6,566,365. See also U.S. Pat. Nos. 6,340,690 and 6,605,614; US Patent Publication Nos. 2002/0099072 and 2003/0225037, as well as International Publication No. WO 01/32153 and WO 00/50424.
BioChem Pharma Inc. (now Shire Biochem, Inc.) also discloses various other 2′-halo, 2′-hydroxy and 2′-alkoxy nucleosides for the treatment of a Flaviviridae infection in US Patent Publication No. 2002/0019363 as well as International Publication No. WO 01/60315 (PCT/CA01/00197; filed Feb. 19, 2001).
ICN Pharmaceuticals, Inc. discloses various nucleoside analogs that are useful in modulating immune response in U.S. Pat. Nos. 6,495,677 and 6,573,248. See also WO 98/16184, WO 01/68663, and WO 02/03997.
U.S. Pat. No. 6,660,721; US Patent Publication Nos. 2003/083307 A1, 2003/008841 A1, and 2004/0110718; as well as International Patent Publication Nos. WO 02/18404; WO 02/100415, WO 02/094289, and WO 04/043159; filed by F. Hoffmann-La Roche AG, discloses various nucleoside analogs for the treatment of HCV RNA replication.
Pharmasset Limited discloses various nucleosides and antimetabolites for the treatment of a variety of viruses, including Flaviviridae, and in particular HCV, in US Patent Publication Nos. 2003/0087873, 2004/0067877, 2004/0082574, 2004/0067877, 2004/002479, 200310225029, and 2002/00555483, as well as International Patent Publication Nos. WO 02/32920, WO 01/79246, WO 0248165, WO 03/068162, WO 03/068164 and WO 2004/013298.
Merck & Co., Inc. and Isis Pharmaceuticals disclose in US Patent Publication No. 2002/0147160, 2004/0072788, 2004/0067901, and 2004/0110717; as well as the corresponding International Patent Publication Nos. WO 02/057425 (PCT/US02/01531; filed Jan. 18, 2002) and WO 02/057287 (PCT/US02/03086; filed Jan. 18, 2002) various nucleosides, and in particular several pyrrolopyrimidine nucleosides, for the treatment of viruses whose replication is dependent upon RNA-dependent RNA polymerase, including Flaviviridae, and in particular HCV. See also WO 2004/000858, WO 2004/003138, WO 2004/007512, and WO 2004/009020.
US Patent Publication No. 2003/028013 A1 as well as International Patent Publication Nos. WO 03/051899, WO 03/061576, WO 03/062255 WO 03/062256, WO 03/062257, and WO 03/061385, filed by Ribapharm, also are directed to the use of certain nucleoside analogs to treat hepatitis C virus.
Genelabs Technologies disclose in US Patent Publication No. 2004/0063658 as well as International Patent Publication Nos. WO 03/093290 and WO 04/028481 various base modified derivatives of nucleosides, including 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides, for the treatment of hepatitis C infection.
Eldrup et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.) p. A75) described the structure activity relationship of 2′-modified nucleosides for inhibition of HCV.
Bhat et al (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.); p A75) describe the synthesis and pharmacokinetic properties of nucleoside analogues as possible inhibitors of HCV RNA replication. The authors report that 2′-modified nucleosides demonstrate potent inhibitory activity in cell-based replicon assays.
Olsen et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.) p A76) also described the effects of the 2′-modified nucleosides on HCV RNA replication.
Drug-resistant variants of viruses can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication, and, for example, in the case of HIV, reverse transcriptase, protease, or DNA polymerase. It has been demonstrated that the efficacy of a drug against viral infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution, or other parameter of the drug can be altered by such combination or alternation therapy. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous pressures on the virus. One cannot predict, however, what mutations will be induced in the viral genome by a given drug, whether the mutation is permanent or transient, or how an infected cell with a mutated viral sequence will respond to therapy with other agents in combination or alternation. This is exacerbated by the fact that there is a paucity of data on the kinetics of drug resistance in long-term cell cultures treated with modern antiviral agents.
In view of the severity of diseases associated with pestiviruses, flaviviruses, and hepatitis C virus, and their pervasiveness in animals and humans, it is an object of the present invention to provide a compound, method and composition for the treatment of a host infected with any member of the family Flaviviridae, including hepatitis C virus.
Further, it is an object of the present invention to provide a compound, method and pharmaceutically-acceptable composition for the prophylaxis and/or treatment of a host, and particularly a human, infected with any member of the family Flaviviridae.
Further, given the rising threat of other Flaviviridae infections, there remains a strong need to provide new effective pharmaceutical agents that have low toxicity to the host.
Therefore, it is an object of the present invention to provide a compound, method and composition for the treatment of a host infected with any member of the family Flaviviridae, including hepatitis C virus, that have low toxicity to the host.
It is another object of the present invention to provide a compound, method and composition generally for the treatment of patients infected with pestiviruses, flaviviruses, or hepaciviruses.